Production of viral interfering substances

ABSTRACT

1. Interferon.

United States Patent I Isaacs et al. 1 Oct. 17, 1972 [541 PRODUCTION OFVIRAL [58] .Field of Search ..424/85 INTERFERING SUBSTANCES V v [72]Inventors: Alick Isaacs, London, England; Jean [56] References citedLindenmann, Gainesville, Fla. OTHER PUBLICATIONS [73] Assignee: NationalResearch Development Burke et 211., British Journal of ExperimentalPatholo- Corporation, London, England gy, Vol. 39, pages 78- 84, Feb.1958. Finter, lnterferons, published by North-Holland [22] Publishing00., 1966, Amsterdam, pages 33, 36, 37, [21] App]. No.: 757,188 233,235, 238, 241,245, 246, and 260.

- Henle et a1., American Journal of Medical Science,

Related 0.8. Application Data VOL 207 page 714 1944 [63] Continuation of575,470 Aug 26 Lindenmann et al., British Journal of Experimental 1966,abandoned, which is a continuation of Pathology page-s 551.; 1957' SerNo 183 413 March 29 1962 abandoned Wolstenholme et al., Interferon,published by Little, which is a continuation-in-part of Ser, No. Brownand Company 1967 Buston pages 210- May 1958 abandoned PrimaryExaminer-Richard L. Huff [30] Foreign Application Priority DataAttorney-Albert Jacobs March 11, 1958 Germany 10 62 391.3 EXEMPLARYCLAIM 1.1 t rf 52 us. Cl ..424/ss e [5 1] Int. Cl 1 6 1 lr 27 0 ll 4Claims, No Drawings PRODUCTION OF VIRAL INTERFERING SUBSTANCES Thisinvention is concerned with the production of viral interferingsubstances.

Viral interference is a phenomenon in which one virus interferes withthe growth of a second virus in living tissues or cells. Thisinterference is not an immunological effect and may occur when theinterfering virus is non-infective.

We have found that during the induction of viral interference bynon-infective virus, a viral interfering substance distinct from thenon-infective virus is produced. The viral interfering substance, whichwe call Interferon, is formed by the interaction of non-infective virusand living cells, and its activity may be recognized by its ability to.inhibit the growth of living viruses.

The production of Interferon is essentially a property of the virus/cellsystem as a whole and, not surprisingly, different virus/cell systemsshow quantitative differences in the yield of Interferon they are ableto produce. It has further been observed that the antiviral substancesproduced in different virus/cell systems exhibit some variation'inbiological properties depending largely on the species of cell in whichthey are produced. Thus, although Interferon exhibits very broadspectrum antiviral activity it is usually found most active when testedin the cells of the same species as the cell in which it was itselfproduced. The degree of specificity shown by Interferon is mostdifficult to interpret in physico-chemical terms because the complexmolecular structure of Interferon is not yet fully understood, and itwill therefore be appreciated that the term Interferon is best regardedas a generic term to be qualified in a particular case to indicate itsorigin, for example, by the use of such terms as monkey kidneyInterferon, chick Interferon, calf kidney Interferon.

The production of Interferon can be explained in the light of thebiological concept of avirulence, a term which eludes definition withabsolute precision but which is well understood by virologists. A viruswhich is fully virulent with respect to a particular species of cellwill enter the cells and cause the cells to replicate more virus bytaking over cellular processes which are normally necessary to maintainthe life of the cell and its reproduction. Virulent viruses thereforecause early death of the cells with the accompaniment of familiarcytopathic effects. Many methods are known of modifying virulent virusesto decrease their infectivity for a particular cell system and theapplication of such methods will give the virus varying degrees ofavirulence. The less inefective, i.e. the more avirulent, a virusbecomes the less its power of reproduction in the cells in which it isincubated, and in parallel with this tendency to reducevirus-multiplication there is an increase in the capacity of the systemto produce Interferon. Consequently, to obtain Interferon mostefficiently virus/cell systems will b used in which the relationship ofthe virus (modified as described hereinafter in further detail) to thecells is one of substantial noninfectivity or high avirulence.

Accordingly, the present invention provides a process for theproducingof a viral interfering substance which comprises incubating avirus in living vertebrate animal cell or tissue material in an aqueousmedium preferably in the presence of oxygen, said material being one forwhich the virus has a sufficiently high degree of avirulencesubstantially to avoid .neutrality are usually employed for theincubation.

The amount of interferon produced depends upon the amount of inactivatedvirus employed, and a second crop can be taken from the living materialon re-incubation with further inactivated virus.

Some degeneration of the cell material will often occur at or near theend of the tissue culture process of this invention, but this is becausein such a system the cell are growing under unnatural conditions andcannot be made to persist indefinitely. It is important, however, todistinguish this type of degeneration which is inherent in the tissueculture method from the cell destruction caused by virulent virus whichmust be avoided if a viral interfering substance is to be produced in asubstantial quantity.

The living animal cell material or tissue in which the virus isincubated so as to liberate Interferon spontaneously into the medium maybe, for example, chick chorio-allantoic membranes, chick embryofibroblasts, monkey kidney cells, rabbit kidney cells, humanamnion orhuman thyroid cells or a cell line from human carcinoma. Each'of theseliving materials has its own optimum cultural conditions as is wellknown in virology. From what has been stated above with regard tospecificity, Interferon prepared in cells of primate origin willunderstandably be preferred for use in the study and understanding ofvirus diseases. However, differences in potency of Interferons fromdifferent sources may often be counterbalanced by the amount of the dosegiven. With regard to the virus, Interferon may be made, for example,from the B/England, Melsess in relation to the cell system frequentlydevelops spontaneously in the course of growing the supply of virus tobe used as inoculum. Again, the procedures of producing the cellmaterial in the most suitable form for subsequent processing ofteninvolve prolonged periods of incubation under conditions which tend toconfer some resistance on the cell themselves.

The inactivation of the virus (especially the myxo viruses mentionedabove) from the infective state to the non-infective state may becarried out by other known methods, for example, by heating or bysubjecting it to ultra-violet light. The inactivating treatment isgenerally such as to abolish or greatly reduce the infectivity of thevirus, whilst retaining its interfering activity. With influenzaviruses, for example, heat treatment at 56C. for 1 hour is sufficientfor this purpose; heating to 60C. would destroy the interfering activityof this virus. Mixtures of heat or ultra-violet inactivated virus andvirus of inherently low virulence may also be used.

The procedure for the production of Interferon will generally be toinoculate the cell material or tissue with the non-infective virus, forexample the Melbourne (1935) strain of influenza virus A, by incubatingthe cell material or tissue for a relatively short time, of the order ofone to three or four hours, in a medium containing the virus. The cellmaterial or tissue is then removed from the medium and washed free of itso that the virus is not carried into the fresh medium in which the cellmaterial or tissue is thereafter incubated. In order to obtain goodyields of Interferon adequate oxygenation of the cell material or tissueis necessary during incubation, which may be carried out in a bufferedsalt solution, forexample Earlessolution (see R. C. Parker, Method ofTissue Culture, 1950). This incubation is carried out, mostadvantageously at.blood heat, for a relatively longer period, forexample overnight, and the Interferon is spontaneously liberated intothe mediumrwith heat-inactivated virus the bulk of the Interferon isliberated between 3 and 12 'hours after commencing incubation, but withultra-violet inactivated virus liberation may continue for 2 or 3 days.

The medium containing Interferon is then separated from the cellmaterial or tissue and may be treated in various ways to purify and/orconcentrate the Interferon. Thus the Interferon may be precipitatedfromthe medium by saturating it with ammonium sulphate. The precipitatemay be dissolved in Earles buffer solution. Ammonium sulphate carriedinto the buffer solution may then be removed therefrom by dialysis.

The Interferon in this solution or in the original medium may bepurified by dialysis against a buffer solution; Interferon is'stable atpH 2 and when so dialysed at this pH some material precipitates leavingInterferon in solution. Interferon is also stable under other pHconditions and is stable for at least two weeks at 2C It is inactivatedby heating for 1 hour at 80C. and pH 7.

The concentration of the Interferon in solution in the original mediummin a fresh solution-after precipitation may be increased by pressuredialysis; through, for example, a Visiting cellulose casing at apressure of 600 mmgllg; the volume of the solution may be reduced50-fold or more .by: this method, leaving Interferon within the dialysissac. F reeze-drying methods can also be used.

Interferon can be distinguished from the original in-.

activated virus by several properties, namely its inability toagglutinate. red blood cells, its resistance to the neutralizing actionof viral antiserum, and its lower sedimentation rate. It is notmeasurably sedimented by centrifuging at 100,000 g. for one-half hour or20,000 g. for 2 hours, although the same treatment removes allinterfering activity from inactivated virus. Interferon is a proteinwith a relatively low weight.

The interfering activity of Interferon is greatly reduced by incubationwith crystalline trypsin or pep- SIII.

The activity of the viral interfering substance may be measured by abiological test and it can be shown in the following way. The substanceis mixed with pieces of chick chorio-allantoic membrane for 24 hours at37C. to allow interference to become established. It is then removed andthe membrane pieces are incubated with live influenza virus. In such anexperiment only a very low yield of virus will result as compared withcontrol pieces of membrane not previously treated with viral interferingsubstance but similarly incubated with live influenza virus. The yieldof virus is conveniently measured by the haemagglutinin titration test.

Interferon is active against viruses which are serologically completelyunrelated to the strain of virus used to produce it. Thus, for example,Interferon inhibitsthe growth of the living viruses mentioned above andalso the haemagglutinating virus of Japan (or Sendai virus) and vacciniaviruses. In the case of the activity of Interferon against vaccinia andother peck-producing viruses, the growth thereof may be estimated by apeck titration method in which the membrane or other living tissue inwhich the virus has been infected are extracted in a grinder and theextract inoculated on the chorion of I 2 tol'3-day developing hens eggsand the. resulting pocks counted after two to three days incubation at35C. Alternatively its activity may be tested directly in fertile henseggs. It is active against cowpox and West Nile Encephalitis virus.Since the discovery of Interferon it has been shown to be effective inthe animal body. Thus inhibition of the. growth of vaccinia in the skinof rabbits and monkeys, inhibition of cowpox virus in the egg,protection of mice against encephalitis virus and protection -of therabbit eye against vaccinia have all been demonstrated.

The antiviral action of Interferon has also. been strikingly observed inman. A number of volunteers received smallpox vaccinations at two sitesin one arm instead of the usual single inoculation after having receivedprevious intraderrnal injections of monkey kidney Interferon and acontrol preparation. Out of 35 cases 23 developed the characteristiclesion on the site that had received the control and no lesion on thesite that had received the Interferon, while 7 cases experienced partialprotection by Interferon as shown by the fact that the lesion developingat the protected site was much smaller than that on the unprotectedsite.

Interferon may be administered topically or parenterally.

The value of the viral interfering substance isthat, whereas vaccinesare not only very specific in their action but do not generally conferimmunity upon the subject. for some two weeks, Interferon is activeagainst a variety of viruses and not only that virus from theinactivated form of which has been used in its preparation, andfurthermore shows its activity in the subject to which it isadministered within a matter of only a few hours.

The invention is illustrated in detail in the following examples. Insome of the examples, increase of the degree of avirulence of the virusfor the cells used is achieved by heat or ultra-violet irradiation; inothers, the virus is inherently of low infectivity for the cells used orbecomes sufficiently avirulent spontaneously under the conditionsdescribed.

EXAMPLE 1 branes were placed in, a flat Roux bottle of capacity 1 literand to this were added 50 ml. of irradiated influenza virus diluted to aconcentration corresponding to 1 ml. of virus-infected egg fluid (l0 tovirus particles) per membrane. The bottle was placed in a horizontalshaking machine delivering 86 to-and-fro strokes to the minute with anamplitude of about 1 inch. This bottle was then incubated at 37C. for 3hours, whereafter the membranes were removed, washed and placed in afresh'bottle with 50 ml. of a buffered salt solution (for example Earlessolution). The bottle was incubated overnight in the shaking machine at37C. and the fluid was removed. 3. Concentration and Purification ofInterferon.

The Interferon may be concentrated and purified in a number of separatesteps which may be applied serially or sequentially, for example:

(a) Interferon was precipitated by saturation with ammonium sulphate,the precipitate was dissolved in Earles buffer solution and the ammoniumsulphate removed by dialysis; (b) Interferon was stable at pH 2 EXAMPLE2.

The inactivated seed virus was prepared by irradiating 3 ml. ofailantoic fluid containing the Melbourne strain of influenza virus in a4 in. Petri dish with a maximum emission at 2,537 A. Thehaemagglutinating capacity of the inactivated virus preparation wasdetermined by making serial two-fold dilutions of this material in 0.9percent NaCl solution in 0.25 ml. volumes and adding an equal volume of0.5 percent fowl red-cell suspension to each dilution. After settling atroom temperature the sedimentation patterns were observed. Oneagglutinating unit'was taken-as the highest dilution giving partialagglutination, and the titres were expressed as the reciprocal of theinitial dilutions of inactivated virus at the end point. The seeding ofthe 5 l. glass bottles containing the membranes was at the rate of l,000haemagglutinating units per membrane.

Eggs were incubated in large commercial incubators for 10 days, and thefertile eggs, after swabbing with 50 percent aqueous ethanol, weredrilled through the end opposite the air sac. After removal of this partof the shell, the embryo, yolk sac and allantoic fluid were pulled outand discarded. The chorio-allantoic membranes were removed and placed ina beaker of Earles medium (40 membranes per beaker). The Earles mediumat all stages were reinforced with 500 ug. of benzylpenicillin/ml. and500 pg. of streptomycin sulphate/ml.

Each membrane was washed individually in more Earles medium to clear itof yolk, albumin and blood, and 40 washed membranes were placed in a5 1. bottle containing 200 ml. of medium. These bottles were seeded withultraviolet irradiated Melbourne virus and rocked in a 37 incubator for3 hours.

After incubation for 3 hours with seed virus, the membranes were removedfrom the bottles, washed in Earle's medium to remove surplus virus andput into further 5 1. bottles, each containing 200 ml. of Earles medium.They were then rocked at 37 for 16-18 hours, when the first harvest wastaken. The membranes were re-incubated as as before in Earles mediumsolution in fresh 5 1. bottles for a second harvest.

The culture fluid containing Interferon was harvested in 500 ml. bloodbottles and clarified by centrifuging at 320 g. for 20 minutes. Thesupernatent Interferon solution was removed and tested for sterility.The second harvest received identical treatment.

' EXAMPLE 3.

Human amnion, monkey kidney (rhesus and cynomolgus) and rabbit kidneyInterferons were made by infecting monolayer cell cultures with theMelbourne strain of influenza virus inactivated with ultraviolet lightas described above for the preparation of chick Interferon. After 3hours at 37C. the virus was replaced by Earles saline which washarvested after 24 hours at 37C. Control preparations were made inthesame way but without infecting the cells with irradiated influenzavirus. The possibility that Interferon wasv produced in such controlcell cultures by the action of a latent virus not producing degenerationof the cultures was not excluded but did not appear-to have been presentto any great extent as judged by the results. Interferon-containingsolutions were dialysed against citrate buffer at pH 2.0 for 24 hoursand then adjusted to pH 7.2.

Concentration of Interferon. Some Interferon preparations wereconcentrated approximately 10 times. This was achieved either byfreeze-drying or by osmodialysis using 40 percent polyethylene glycol(Carbowax 20,000 M or 4,000 M, Union Carbide Co.). Polyethylene glycol,especially the 4,000 average molecular weight material, diffused throughthe dialysis bags into the Interferon, but did not interfere with themeasurement of Interferon either in vitro or in vivo.

against the growth of vaccinia in the rabbit skin.

EXAMPLE 4.

Calf kidney cells were grown on six sides of Pyrex babiesfeedingbottles, which were rotated in an incubator room maintained at 37C. Whenconfluent sheets of cells had been obtained, the serum-containing mediumused to grow the cells was drained, and the cell sheets washed withphosphate buffered saline solution. Each bottle was inoculated with 20ml. of a dilution of l/ 10,000 in medium 199 of the Kunz strain ofinfluenza A virus (which was stored in the form of high titre allantoicfluid virus). The bottles were incubated with rotation for a further36-60 hours. The rate of growth of virus was followed by measuring therate of formation of haemagglutinins. Approximately 12 hours after thetime when the peak of haemagglutin formation had been reached, thebottles were removed from the incubator. The fluids from groups of 30bottles were drained, pooled together and tested for freedom frombacterial and other contaminants. When these tests had been completed,all the pools were combined and passed through a porosity 3 sinteredglass filter to remove cell debris. N/ l0 hydrochloric acid was added tobring the pH down to a level of 2. This degree of acidity was maintainedfor 24 hours, after which N/ 10 EXAMPLE 5.

Tissue cultures were prepared in Roux bottles from cortical tissueisolated from the kidneys of monkeys. This tissue was cut into smallpieces and suspended in medium 199 (Morgan et al., Proc. Soc. Exper.Biol. & Med. 73,..l-8) anddigested at 37C. with 0.25 percent trypsin for1 hour. The cells were then washed by centrifugation in fresh medium 199and suspended for the growth phase in medium 199 containing 2 percenthorse serum. After seven days incubation confluent first generationmonolayer cultures were obtained. Each confluent culture was fluidchanged withv 100 ml. of medium 199 containing 2 percent NaHCO Kunzinfluenza virus was grown in fertile hens eggs for 2 days and theallantoic fluid was used to seed the tissue cultures, l ml. of the virusfluid, which contained 4,000 haemagglutinating units being added to eachRoux bottle. The cultures were incubated at 37C. for 3 days when thefluidswere harvested and adjusted to pH 2.0 by the addition of N/l HCl(approximately 40 ml/litre). After three hours N/l NaOl-l was added tobring the pH to 7.0. Haemagglutinin tests were negative. The fluids weredialysed against distilled water for 24 hours to lower the saltconcentration. Gelatin was then added to give a concentration of 0.1percent and the material freeze-dried.

EXAMPLE 6.

An inactivated seed virus was prepared by irradiating 3 ml. of allantoicfluid containing Kunz strain of influenza virus in a 4 in. Petri dishfor l min., 7 cm. beneath a Phillips U.V. tube with a maximum emissionat 2,537 A. The haemagglutinating capacity of the inactivated viruspreparation was ,determined,'and the fluid was used to seed monolayertissue cultures obtained. as in Example at the rate of 12,000haemagglutinating units per Roux bottle. After 48 hours incubation at37C. the fluids were harvested and dialysed against distilled water asin Example 1.

EXAMPLE 7.

The inactivated seed virus was prepared by the method given in Example6.

The chorio-allantoic membranes were removed from fertile hens eggs whichhad been incubated in a commercial incubator for 10 days. The membraneswere washed and placed in 5 literPovitsky bottles, (40 membranes in eachbottle) containing 200 ml. of Earles medium fortified with 500 1g. ofbenzyl-penicillin/ml. and 500 pg. of streptomycin sulphate/ml. The seedvirus was added at the rate of 1,000 haemagglutinating units permembrane, and the bottles were incubated at 37C. for 3 hours. Themembranes were removed and washed in Earless medium and transferred to 5liter bottles containing 200 ml.of the same medium with added penicillinand streptomycin and rocked at 37C.

for 18 hours. The first fluid harvest was then taken and the membraneswere reincubated as before for a second harvest. The culture fluidcontaining Interferon was clarified by centrifugation and concentratedby osmodialysis against polyethylene glycol.

EXAMPLE 8.

The method used was basically that described in Example 7 but allantoicfluid containing A/Singapore inflenza virus was used to seed the chickmembranes. After incubation for 48 hours at 37C. the fluids wereharvested, acidified to pH 2 with N/ 1 NC] for 3 hours to destroy thelive virus and then neutralized with N/l N aOl-l This Interferonsolutionv was subsequently clarified by centrifugation, tested forsterility and concentrated by osmodialysis.

EXAMPLE 9.

B/England strain of influenza virus was grown in fertile hens eggs andthe allantoic fluid containing-the virus used in the production ofInterferon in hens eggs. The haemagglutinating titre of the allantoicfluid containing the virus was .1 :4,000 per ml. 0.1 ml. portions of 10'dilution were inoculated into the allantoic cavities of fertile henseggs which had been incubated in a commercial incubator for 10 days. Theeggs were then further incubated at 37C. for 2 days. The. allantoicfluid was then harvested, clarified by centrifugation and treated at pH2 for 3 hours to inactivate the virus. After returning the reaction topH 7.0 the Interferon was concentrated by freeze-drying.

EXAMPLE 10.

Melbourne strain influenza virus was inactivated as described in Example6 and used to inoculate hens eggs as described in Example 9. The eggswere incubated for 2 days at 37C. The allantoic fluid was thenharvested, clarified and concentrated.

\ EXAMPLE 1 l.

l. The Kunz strain of influenza A virus was grown in fertile hens eggs.The allantoic fluid was diluted with medium 199 to contain 200-500haemagglutination units of virus/ml.

2. Tissue Trypsinised rhesus monkey kidney tissue was added to nutrientmedium consisting of 0.5 percent lactalbumen hydrolysate and 5 percenthorse serum in Earles saline contained in flat-sided bottles of about520 ml. capacity, each bottle containing about 5 X 10 cells in ml. ofmedium. The bottles were incubated for 56 days at37C. while a cell sheetdeveloped. The nutrient medium was then decanted and the cell sheet waswashed with an isotonic phosphate-buffered saline, PH 7.5.

3. Formation of Interferon. Each bottle received 0.5

ml. of the virus in medium 199, and a further 50 ml. of medium 199. Thebottle containing the cell sheet and the virus was incubated for 3 daysat 37C. Some bottles received only 50 ml. of medium and no virus, andthese served as controls.

4. Recovery and assay of Interferon. The supernatant containinglnterferon was decanted from the cell sheet and tested for sterility andfreedom from mycobacteria. Residual virus was inactivated by bringingthe liquid to pH 2, storing for 24 hours at 4C. and bringing back to pH7. The solution of Interferon was also submitted to EXAMPLE 12.

Human thyroid cell cultures were prepared by mincing surgical specimensfrom cases of goitre, trypsinising overnight at 4C. in 0.25 percentDifco trypsin and growing the cells in medium 199 containing 10 percentcalf serum. The agar overlay medium was composed of Earles balanced saltsolution containing Eagles nutrients (Eagle, 1955), 0.5 percentlactalbumin.

hydrolysate, percent skim milk, l:30,000 neutral red, and 1.5 percentNoble's agar. All media contained 100 units/ml. of penicillin andstreptomycin. Interferon was prepared by inoculation of these cultureswith a 10 dilution of Newcastle disease virus and incubation at 36C. for2 days. At this time the cells appeared normal and the fluids wereharvested and treated at pH 2 in order to destroy residual virus. The pHwas lowered to 2 (red color of thymol blue) by adding l/3 N hydrochloricacid in the presence of l/ 100,000 thymol blue. The acidified fluid waspoured into a fresh vessel and the pH raised to 7.8 (blue color ofthymol blue) with l/3 N sodium hydroxide. The product was an aqueoussolution of Interferon.

What is claimed is: l. Interferon. 2. Human interferon. 3. Monkeyinterferon. 4. Chick interferon.

* l I F t

1. INTERFERON.
 2. Human interferon.
 3. Monkey interferon.
 4. Chickinterferon.